Cell spraying device, method and sprayed cell suspension

ABSTRACT

The invention provides a device and methods suitable for producing a cellular spray of cells. The sprayed cells are of interest for covering and growing on a surface, including a skin wound. In applying the method and/or using the device, cells for grafting onto a patient are dispersed in a solution and sprayed with the device for distribution over the recipient&#39;s graft site.

FIELD OF THE INVENTION

This invention relates to a simple, rapid, and cost effective techniquefor the grafting of cells, in particular to a device for spraying a cellsuspension from a tissue sample obtained from a donor site anddistributing that cell suspension to a recipient site.

BACKGROUND

Spraying of cells may be of interest for the distribution of cellsuspensions onto a tissue wound. This can be applied, e.g., in generalsurgery to help regenerate tissue trauma. There are many methods fortreating skin wounds known to those skilled in the art. For example,skin grafting techniques exist, which aim to reconstruct skin areas ofthe body that have suffered either damage or defects to the skin. Ingeneral, these types of grafts are classified according to theirhost-donor relationship and by their thickness. The most clinicallyapplied graft is the autologous graft, whereby tissue is taken from onearea of the body and applied to another area. The grafted tissue thendevelops a new blood supply and attaches to the underlying tissues.There are several types of skin grafts presently used, includingsplit-thickness, full-thickness grafts, and micro-grafting. Each ofthese graft types must be prepared using certain techniques, and eachone has its inherent advantages and disadvantages. Split-thicknessgrafts often require considerable skill, time and expensive equipment.Further, donor sites are painful, result in scarring and limit thecoverable area. Although split-thickness grafts may be more successfulthan full-thickness grafts, they are usually cosmetically lessattractive. Full-thickness grafts require less skill or expensiveequipment, and their cosmetic appearance is better than that ofsplit-thickness grafts. However, full-thickness grafts do not “take” aswell as split-thickness grafts. Micro-grafts are more easilyaccomplished and require no special instruments. However, their cosmeticappearance is not as good as other techniques, as the resulting scarringis unacceptable.

A variation to the above grafting techniques is the mesh graft, which isa type of split-thickness or full-thickness skin graft in which parallelrows of slits are cut into the treated tissue. Some of the advantages ofmesh grafts include: greater coverage of the effected area, drainage ofblood or serum from beneath the graft, and increased conformity of thegraft to uneven recipient areas. This technique has been verysuccessful, with 90 to 100 percent “take” after the grafts have beenapplied on healthy granulation beds.

An alternative to split-skin grafting is to form a blister under suctionat a donor site, then remove the skin above the blister and transplantit onto the recipient site. The production of blisters to treat woundshas been used since the 1960s. The blisters are produced by a suctiondevice, such as Dermavac™, at a suction pressure of approximately250-300 mmHg for 1-2 hours. The blisters are then cut off and placed onthe wound. The healing time is around 10-14 days. There are severaldisadvantages to this method such as the amount of time required toprepare the graft is too long and the graft may not result inre-pigmentation of the area; or uneven pigmentation is common around theedges of the area of treatment.

Micro-grafting has become a more common approach for large area coverand involves the “snipping off” of a number of very small sections oftissue from a donor site and applying them to a dressing that is whichis in turn applied to the wound area.

Another technology for the generation of tissue in vitro is to cultureepidermis. Cultured epithelial autografts (CEA), provided in confluentgrown cell sheets, are an important adjunct in the coverage of burns andother situations in which large areas of the body's surface experienceskin loss. There are many centres throughout the world with tissueculture facilities whose aim is to produce autologous epithelial graftsfor use in a wide variety of applications. The usefulness andapplication of CEA is related to its ability to achieve confluent cellssheets suitable for grafting. This technique overcomes many of thedisadvantages of the previous treatments described above. For example,cultured epithelial autografts reduce the demand for donor sites.However, these autografts are slow growing and require time to cultur,which often exceeds the preparation time of the recipient's sites.Moreover, blister formation by wound secretion below the sheet graftshinder grafting.

Navarro et al. (2000) and Wood et al. (2003) describe the use of singlecells suspended in Hartmans's solution and distributed over the wound,thus avoiding the sheets. The cell suspension may be delivered via apipette, common “eye-droppers,” syringe and needle, and/or other similardevices to place small quantities of cellular suspension on a graftsite. As method of choice a mechanical hand driven spray technique isdescribed (see references).

The spray technique adresses some afore mentioned problems in the field.A hand driven spray method and subsequently the distribution of thecells, however, is not performed in a controlled manner and results inuneven cell distribution.

The present invention provides a method, a device, and a cell suspensiongenerated by using the method, each of which seeks to ameliorate some ofthe disadvantages associated with prior art CEA grafting technology.

SUMMARY OF THE INVENTION

The present invention provides a method and/or device suitable forproducing a transplantable cellular spray of living cells suitable forgrafting to a patient. In applying the method and/or in using thedevice, cells suitable for grafting to a patient are dispersed in asolution and sprayed with the device for distribution over the recipientgraft site.

According to the invention a method is provided for spraying a cellsuspension through a controlled spray head suitable for application to apatient utilizing a spray device, which method comprises the steps of:(a) subjecting a tissue sample including cells suitable for grafting toa patient, to at least a physical and/or chemical dissociating meanscapable of dissociating cells in the tissue sample; (b) taking the cellssuitable for grafting on to a patient into a saline solution, (c)filtering the cellular suspension produced to remove large cellularconglomerates; and spraying the cell suspension through a spray head.

According to the invention an electronically controlled apparatus isprovided as a medical device for distribution of tissue regeneratingcells in a sterile suspension over a tissue surface via electroniccontrolled compressed gas and/or pump driven spraying through asterilizeable spray head, providing continuous force application in asingle shot and generating suspension drops containing cells.

According to the invention there is provided a cell suspension producedaccording to the above-described method. Preferably the cells in thesuspension are autologous cells (i.e. they are isolated from the patientrequiring an autograft), or stem cells.

According to another aspect of the invention a method is provided totreat a patient in need of graft surgery.

Other aspects and advantages of the invention will become apparent tothose skilled in the art from a review of the ensuing description, whichproceeds with reference to the following descriptions and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 compares two application modes of skin cells to a patient.Application of the method and/ or device described in this text at hand,spraying skin cells onto a skin wound surface, is illustrated on theleft side. This can be compared to the state of the art medicaltreatment with skin cell application using confluently grownkeratinocyte sheets (right). Using sprayed cells result in the need offewer cells while in a larger treatment surface can be enabled fortherapy. Blister formation is avoided by the use of single cells withoutforming a closed sheet. Reducing the cell number speeds up applicationtime by avoiding an in vitro cell expansion. This reduces in vitrodifferentiation and therefore better preserves basal keratinoyteprogenitor cells in the cell suspension.

DESCRIPTION OF THE INVENTION

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variation and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification. Functionally equivalent products, compositions andwhere appropriate methods are clearly within the scope of the inventionas described herein.

Throughout this specification and the claims that follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

Having regard to the above, this invention provides a unique methodand/or device suitable for producing a transplantable cellularsuspension of living tissue suitable for grafting to a patient. Inapplying the method and/or in using the device cell preparations ofdifferent origin may be used. This includes stem cell preparations andpatient autologous cells, whereas donor tissue is harvested andsubjected to a tissue dissociating means. Cells suitable for grafting toa patient, or back to a patient, are dispersed in a solution that issuitable for immediate dispersion over the recipient graft site.

The subject invention has many advantages over the prior art some ofwhich are described in the following paragraphs.

1. It provides a time efficient method for supplying a cellular cover toa tissue in a clinical setting. That is, cells are finely and evenlydistributed over a wound, avoiding the use of cell sheets. This isachievable because there is a controlled procuring of the cellsuspension with a method provided by an apparatus, thus allowing cellspraying to be performed more evenly than the mechanical hand operatedmethods of the prior state of the art.

2. It provides a method and an apparatus, which avoids the blisterformation associated with the use of conventional CEA's.

3. It aids in the achievement of rapid cell coverage in areas of tissuewounds, tissue trauma/injury and donor sites. It provides a means forreducing the size of skin cell donor sites—the biopsy donor site ismarkedly smaller than a split skin graft donor site and reduces oreliminates the use of split skin graft donor sites; improves theexpansion rate of cell coverage; improves the rate of healing of smallburns; is useful for small areas of skin reconstructions, such as scars;and improves scar quality.

4. It provides a means for the treatment of various skin disorders ordiseases. For example, it may be used for the following: epidermalresurfacing, replacement after skin loss, site match-up duringre-pigmentation of an area of skin, treatment of burn wounds,leukoderma, vitiligo, piebaldism, in the treatment of scars—for example,caused through incorrect wound healing, improper scar direction or scardistortion from wound contraction, acne scars; resurfacing cosmeticdermabrasion, resurfacing after laser treatment and in association withdermal reconstruction. Additionally the method may be used for cellreplacement therapy, including, for example, nerve cell replacementtreatment, epithelial cell (such as urothelial cell, buccal mucosal celland respiratory epithelial cell) replacement treatment, endothelial cellreplacement treatment and osteogenic precursor cell replacementtreatment. The method may also be used to stimulate tissue regenerationin surgically induced wounds.

5. It provides a means to produce a suspension of cells in a ratio toeach other comparable with those seen in situ. That is, due to themanner of preparation of the cellular suspension, cells such askeratinocyte basal cells, Langerhans cells, fibroblasts and melanocytestypically have enhanced survival rates in comparison to standard tissueculture techniques, whereby selective cell culture can result in theloss of certain cell types. This has the advantage of allowing for thecorrect re-pigmentation of skin after a skin graft.

7. It allows faster surgery and healing—thereby reducing trauma forpatients during the phase of their medical care.

The invention relates to at least two distinct cell sources, allsuitable for use in resurfacing and regeneration of damaged tissue: (i)non-autologous cells, including stem cells, and (ii) autologous cells,including the patient's own progenitor cells.

The invention provides a method for preparing an autologous cellsuspension. According to this method, tissue is harvested from a patientby means known in the art of tissue grafting. Preferably this isachieved by taking a tissue biopsy. With the harvesting of the biopsyconsideration must be given to the depth of the biopsy and size of thesurface area. The depth and size of the biopsy influence the ease atwhich the procedure can be undertaken and the speed with which a patientrecovers from the procedure. In a highly preferred form of the inventionthe chosen donor site should appropriately match the recipient site, forexample post-auricular for head and neck, thigh for lower limbs,inner-upper-arm for upper limbs, or palm for sole or vice-versa.

Once a biopsy has been harvested from a patient the tissue sample issubjected to physical and/or chemical dissociating means capable ofdissociating cellular stratum in the tissue sample. Methods fordissociating cellular layers within the tissues are well known in thefield. For example, the dissociating means may be either a physical or achemical disruption. Physical dissociation means might include, forexample, scraping the tissue sample with a scalpel, mincing the tissue,physically cutting the layers apart, or perfusing the tissue. Chemicaldissociation means might include, for example, digestion with enzymessuch as trypsin, dispase, collagenase, trypsin-edta, thermolysin,pronase, hyaluronidase, elastase, papain and pancreatin. Non-enzymaticsolutions for the dissociation of tissue can also be used. Preferably,dissociation of the tissue sample is achieved by placing the sample in apre-warmed enzyme solution containing an amount of enzyme sufficient todissociate cellular stratum in the tissue sample.

After the tissue sample has been immersed in the enzyme solution for anappropriate amount of time, the sample is removed and washed withnutrient solution.

The saline/nutrient solution used in the method should be capable ofsignificantly reducing and more preferably removing the effect of theenzyme either by dilution or neutralization. The nutrient solution usedin the method will also preferably have the characteristics of being (i)free of at least xenogenic serum, (ii) capable of maintaining theviability of the cells until applied to a patient, and (iii) suitablefor direct application to a region on a patient undergoing tissuegrafting. After application of a suitable saline/nutrition solution tothe tissue sample, the cellular stratum of the sample is separatedpermitting the cells capable of reproduction to be removed from thecellular material and suspended in the nutrient solution. Where thetissue sample is skin, the dermis and epidermis are preferably separatedto allow access to the dermal-epithelial junction of both surfaces.

Cells capable of reproduction are then removed from the separatedstratum by any means known in the art. Preferably, the reproductivecells are scraped off the surface of the stratum using an instrumentsuch as a scalpel. Cells capable of reproduction within thedermal-epithelial junction include but are not limited to keratinocytebasal cells, Langerhans cells, fibroblasts and melanocytes. Followingrelease of the cells from the tissue sample they are suspended in thesaline/nutrient solution.

The invention provides simultaneously a method for using anon-autologous cell suspension. To procure cells of any source, thecells are suspended in an aquaeus saline/nutrition solution. Thesolution may be anything physiological from a basic salt solution to amore complex nutrient solution. Preferably, the nutrient solution isfree of all serum but contains various salts that resemble thesubstances found in body fluids; this type of solution is often calledphysiological saline. Phosphate or other non-toxic substances may alsobuffer the solution in order to maintain the pH at approximatephysiological levels. Suitable nutrient solutions that are preferredbase on Ringer-lactate solutions, including Hartmann's solution,dialysis solutions, and on peripheral intravenous nurtition solutions.

Preferably only a small volume of solution is applied to the tissuesample after the harvesting steps, or by suspending non-autologouscells, otherwise the suspension may become too fluid therein providingdifficulties in applying the suspension to the graft.

The cell suspension is then applied by using the spray device, describedin the claims. To avoid excessively large cellular congregates in thecellular suspension the suspension is preferably filtered, either priorto using the suspension with the device, or by a specific feature of thedevice.

Prior to application with the device or immediately after filtering, thecellular suspension may be diluted to produce an appropriate celldensity suitable for the purpose with which the suspension is to beused.

According to the invention there is provided a sprayed aqueous cellsuspension, highly suitable for tissue regeneration and graftingtechniques, produced by the method described. An important advantage ofthe invention is an even cell distribution.

An important aspect of utilizing such a suspension in graftingtechnology is that it can be used to greatly expand the area or volumeof a wound that can be treated quickly by in situ multiplication of alimited number of cells. Cellular multiplication is encouraged on thepatient rather than in an in vitro system, as provided by the state ofthe art CEA method.

The number and concentration of cells seeded onto graft site may bevaried by modifying the concentration of cells in suspension, or bymodifying the quantity of suspension that is distributed onto a givenarea or volume of the graft site.

Another unique feature of the cell suspension produced according to themethod of the invention is that the composition of cells in the cellularpreparation is comparable to that seen in situ compared to prior art CEAcellular preparation. Importantly, it contains the basal keratinocytesand skin progenitor cells for skin regeneration, which are typicallylost in the CEA method. In this prior art, culture of the cellularpreparation utilizes selective culture for keratinocytes, therefore theloss of cellular constituents such as skin progenitor cells, fibroblastsand melanocytes occurs, whereas the cellular suspension produced by themethod of the invention has a cell composition comparable to the in situcell population.

According to a further aspect of the invention there is provided amethod of treatment of the patient requiring a tissue graft. By thismethod the cellular suspension produced according to the invention isapplied to a graft site.

According to the invention there is provided an apparatus containing aspray head to distribute the cells. The suspension may be sprayedthrough any type of nozzle that transforms liquid into small airbornedroplets.

According to the invention there is provided an electronicallycontrolled apparatus as a medical device to operate the spraying througha sterilizeable spray head. Preferrably the apparatus enables adistribution of cells using a 0.5-60±20 ml sterile cell suspensionthrough a spray head. Preferrably, the apparatus transfers the cellsuspension from a medical grade disposable sterilizeable syringe,including 0.5-60 ml sterile Luer-lock syringes.

The apparatus can be operated preferrably basing on producing compressedgas, e.g. air, for the spray head, or forcing the cell suspension pumpdriven through the nozzle, e.g. by motor operated pushing of a sterileLuer-lock syringe containing the cell suspension, without mixing withgas. The apparatus preferrably provides continuous force applicationover a range of 0.5-10±1.0 minutes for in a single shot, or severalshots, and generates suspension drops containing cells in the range of30-500+200 millimeter.

The apparatus may provide means to measure and control parameters suchas flow, pressure, and/or temperature.

The apparatus preferrably also transfers the cell suspension from amedical grade sterilizeable container to the sterilizeable spray headvia a disposable filter capable of separating large cellular congregatesfrom a cellular suspension. Any filter capable of separating excessivelylarge cellular congregates from the suspension may be used. In a highlypreferred form of the invention the filter exibits a cut off ofapproximately 5-100 cells, preferably 20-60 cells and most preferred 40cells.

The apparatus may comprise a first and second member wherein: (i) thefirst member includes power supply, gas/air supply and electroniccontrols, and (ii) the second member includes a sterilizeable spray headand the container with the cell suspension. In that case both membersare connected through a cable/wire/tube sensor/effector connector whichmay be sterilizeable or can be covered with a sterile operation foilhose and has suitable connectors to the memebr (i) and (ii).

The apparatus may comprise a first and second member wherein bothmembers are wirelessly connected for data exchange, including blue toothtechnology, to connect sensor/effector controls in the first and secondmember.

The apparatus may also feature battery operation, facilitating an easyuse in operation theaters. In that preferred case, the apparatuscomprises an all-in one device for hand-held operation.

After the cell suspension has been applied to the recipient graft site,the wound may be covered with a wound dressing. Preferably, the healingof the wound is followed up by standard protocols for graft treatmentknown to those skilled in the art.

EXAMPLE

If not otherwise indicated, all materials were purchased from BiochromAG, Berlin, Germany. Media were supplemented with antibiotics(Penicillin/Streptomycin, 120 μ/g/ml) and antimycotics (Amphotericin B,2.5 μg/ml). A 1 cm² skin biopsy was obtained after obtaining informedconsent of the donor and cut into 2 mm² pieces. The method is describedin more detail in Johnen C, et al., Burns. 2006; 32(2). Prior toseparation of epidermis and dermis the pieces were exposed to 0.4%collagenase (Serva Electrophoresis GmbH, Heidelberg, Germany) in DMEM at37° C. Separated epidermis was incubated with 0.05% trypsin/0.02%EDTA-solution for 15 minutes. The single cell suspension was cultivatedin a standard culture flask with serum free culture medium (EpiLife,TEBU, Offenbach, Germany). Cells were incubated at a cell density of 10⁴per cm², using a CO₂-incubator (Heraeus BB 6060, Kendro, Langenselbold,Germany) at 37° C. in a humidified atmosphere with 5% CO₂. Medium waschanged every two days. As 80% confluence was reached, cells weredetached by trypsinization and used with the above described compressoroperated spray device prototype. Operation partameter were set to an airflow of 3.7 l/min and a fluid flow of 4.2 cc/min. This adjustmentresulted in a spray pressure of 8.2 mmHg. The cells were sprayed into anon medium filled standard cell culture dish at a density of 10⁴ cellsper cm². As control cells from the same suspension were cultivated,under the above described culture conditions after pipetting into amedium filled culture flask with the same density. Cell morphology wasmonitored by light microscopy (Zeiss, Axiovert 25). Sprayed andnon-sprayed cells showed similar morphologic appearance in light- andphase-contrast microscopy, they also showed comparable follow up culturebehavior.

Further examples will be given in a manuscript, to be submitted afterfiling of the application at hand to Elsevier, N.Y., USA forconsideration in the book “Principles of Regenerative Medicine” editedby Antony Atala et al., to be published in 2007.

Modifications and variations of the described methods and device of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the relevant field in which this invention resides areintended to be within the scope of the described claims.

1. A device for an electronically flow controlled distribution of tissueregenerating cells, including skin cells, in an approximately 0.5-80 ml,preferably 5-40 ml and most preferably 5-20 ml sterile suspension acrossan area for further growth, via controlled compressed gas and/or pumpdriven spraying through an electronically pressure and/or flowcontrolled spray head, providing continuous application over a range ofapproximately 0.5-10, preferably 1-5 and most preferably 1-2 minutes ina single shot, or several shots, preferably up to 3 shots, whilegenerating suspension drops containing cells ranging between 30-500±200mm in size, wherin the spray head may be sterilizeable or can be coveredwith a sterile surgery foil hose, and wherein the following proceduresare enabled: (a) subjecting a tissue sample including cells for graftingonto a patient, to at least a physical and/or chemical dissociatingmeans capable of dissociating cells in the tissue sample; (b) taking thecells, which may also have been expanded after the aforementioned step,for spraying into a saline solution, wherein the solution is (i) free ofxenogenic serum, (ii) capable of maintaining the the cells alive untilapplied, (iii) contains electrolytes in a physiologic composition, and(iv) allows direct application to a biomaterial surface or region on apatient undergoing tissue grafting; (c) filtering the cellularsuspension produced according to steps (a) and (b) to remove cellularconglomerates of more than 10-500 cells, preferably 50-300 cells andmost preferably 50-100 cells; and (d) distributing the cell suspensionthrough the flow controlled spray head onto the receipient surface.
 2. Adevice according to claim 1 that is driven via a gas compressor.
 3. Adevice according to claim 1 that is driven via a motor.
 4. A deviceaccording to claims 1 to 3, which contains sensors to measure flowand/or pressure, and/or temperature.
 5. A device according to claims 1to 4, which contains sensors to measure and feedback controls to controlflow and/or pressure, and/or temperature.
 6. A device according toclaims 1 to 5, which transfers the cell suspension from medical-gradedisposable sterilizeable syringes, including 0.5-60 ml sterile Luer-locksyringes, preferably 1-50 ml and most preferred 5-20 ml syringes.
 7. Adevice according to claims 1 to 6, which transfers the cell suspensionfrom a medical-grade sterilizeable container, including luer-locksyringes, to the sterilizeable spray head via a disposable filtercapable of separating large cellular congregates with a cut off ofapproximately 5-100 cells, preferably 20-60 cells and most preferred 40cells from a cellular suspension.
 8. A device according to claims 1 to7, which is battery operated.
 9. A device according to claims 1 to 8 fordistributing a cell suspension, containing a first and second componentwherein: (i) the first component includes the power supply, gas/airsupply and electronic controls, and (ii) the second component includes asterilizeable spray head and the container with the cell suspension; andwherein both components are connected through a cable/wire/tubesensor/effector connector, which may be sterilizeable or can be coveredwith a sterile operation foil hose and has suitable connectors to thecomponents (i) and (ii).
 10. A device according to claim 1 to 9 fordistributing a cell suspension, containing a first and second componentwherein both components are wirelessly connected for data exchange,including blue tooth technology to connect sensor/effector controls inthe first and second component.
 11. A device according to claims 1 to 10which utilizes a solution as an aquaeous solution containingelectrolytes in a physiologic composition, including Ringer-Lactate likeelectrolyte solutions, including Hartman's solution.
 12. A cellsuspension produced according to the devices and methods of claims 1 to11.
 13. A cell suspension produced according to at least one of theaforementioned claims prepared from autologous cells, including in vitroexpanded autologous cells.
 14. A cell suspension produced according toat least one of the aforementioned claims prepared from stem cells. 15.A method of utilizing the device according to at least one of theaforementioned claims and producing a cell suspension for treating apatient in need of graft surgery, which is is comprised of the steps:(a) preparing a cell suspension according to the method of claims 1 to14; and (b) administering the suspension directly to a region on thepatient that requires a cell graft in a manner that facilitates sprayingof the cell suspension in an even distribution over the graft area. 16.A method of utilizing the device according to at least one of theaforementioned claims and producing a cell suspension for treating apatient in need of graft surgery with a cell suspension according to oneof the aforementioned claims, said devices according to claims 1 to 11and administering the suspension to a region on the patient thatrequires a cell graft in a manner that facilitates spraying of the cellsuspension in an even distribution over the graft area.
 17. A method ofcoating an artificial surface or a biomaterial surface for commercialuse with the device according to at least one aforementioned claims,said method is comprised of the steps: (a) preparing a cell suspensionaccording to the method of claims 1 to 16; and (b) administering thesuspension directly onto an artificial or a biomaterial that requires acell coated surface in a manner that facilitates spraying of the cellsuspension in an even distribution.